ErbB receptor tyrosine kinases play essential physiological roles in mediating cell proliferation, differentiation, survival, migration and differentiation during development and tissue homeostasis, and their overexpression and/or hyperactivity are directly linked to oncogenesis in a number of human cancers. ErbB receptor activation also initiates cytoprotective signaling that limits the extent of tumor killing by radiation and/or chemotherapy, and their inhibition represents an important strategy for radio- and chemo-sensitization of tumors. Elucidating novel aspects of ErbB receptor signaling is therefore a major goal in cancer biology. Ligand binding to ErbB receptors induces rapid intemalization into endosomes followed by a sorting process to target them to lysosomal degradation or for recycling. Rather unexpectedly, recent studies have shown that activated ErbB receptors continue to signal after their internalization. The nature of the signaling endosomal compartment(s), the specific signaling events that take place in endosomes, and the cellular mechanisms that regulate the delivery of activated ErbB receptors into these signaling compartments remain unknown. A number of experimental findings lead us to postulate a novel role for Src tyrosine kinase as a positive regulator and/or mediator of ErbB signaling within the endosomal compartment. To test our hypotheses, we will compare immortalized, non-tumorigenic human mammary epithelial cells (MECs) with their isogenic derivatives overexpressing EGFR or ErbB2 with or without Src, which models the co-overexpression of these tyrosine kinases in breast and other cancers. We will first establish that Src is a positive regulator of ErbB receptor signals and biological responses in MECs. We will then use two-color imaging and biochemical analyses to determine the colocalization of Src and ErbB2 within the endocytic pathway, and identify the specific compartment in which they reside. We will carry out analyses to assess if endosomal ErbB receptor-Src complexes signal and determine the nature of such signals and their biological consequences, using Src kinase inhibition and perturbations of transport within the endocytic compartments. Validation of our hypotheses will represent a major shift in the current paradigm of ErbB receptor signaling, and will provide novel biochemical insights into the role of Src overexpression together with ErbB receptors in breast and other epithelial cancers. Our studies may identify novel targets for therapeutic intervention relevant to ErbBand Src-overexpressing human cancers.